Cancer is driven by somatically acquired point mutations resulting from DNA nucleotide damage, and genomic rearrangements arising from DNA double strand breaks (DSBs). The etiology of these DNA lesions remains unclear, but it is known that cellular processes that increase rates of DNA damage and frequencies of DSBs can induce lesions that initiate transformation. Mutations and genomic rearrangements that develop in a malignancy may also result in clonal populations that are either resistant or sensitive to particular chemotherapeutic agents. Identifying cellular enzymes capable of generating DNA lesions that drive malignant progression will suggest targets for development of novel drugs to treat therapy-resistant cancers. The human genome encodes seven APOBEC3 (hA3) enzymes that normally induce mutations and DSBs. These hA3 enzymes, and the related AID protein, constitute a family of deaminases that convert cytosines to uracils in single-stranded DNA substrates. The APOBEC3 proteins (hA3A through hA3H) mutate viral genomes and thereby confer antiviral functions. AID induces mutations and DSBs in immunoglobulin loci in B lymphocytes to generate antibody diversity and thereby protect humans from pathogens. Accumulating evidence suggests that off-target APOBEC3/AID activities induce mutations and genomic rearrangements that drive initiation, progression, and drug-resistance of tumors, but may also present opportunities for new cancer treatment strategies. The objectives of this proposal are to assess the impact of hA3 expression on (i) proliferation, genome integrity, and malignant transformation in mouse models and (ii) acquisition of drug resistance and sensitivity in human cancer cells. We focus on hA3A and hA3B since these active cytosine deaminases have been shown to cause mutations and DSBs in the genome when over-expressed in human cells. The hA3A protein is expressed in hematopoietic cells and can be induced to high levels by virus infection and interferon, while hA3B is broadly expressed but is upregulated in a number of human cancers. Our hypothesis is that sustained expression of hA3A or hA3B results in deamination of the host cellular genome, inducing a cancer- promoting mutator phenotype and/or development of drug-resistant cancers. We will test our hypothesis by assessing the impact of hA3A and hA3B expression on mouse hematopoietic cells (Aim 1) and the ability of hA3A and hA3B to cause drug-resistant cancer cells, yet also render cancer cells sensitive to inhibition of DNA repair factors (Aim 2). The rationale for the proposed studies is that individual expression of hA3A or hA3B in cells that lack endogenous protein will reveal insights into how hA3 proteins contribute to initiation and drug- resistance of human cancers. The outcomes of these Aims will reveal the extent to which hA3A and hA3B confer pro-oncogenic activities in tumor initiation, progression, or acquisition of therapy resistance, and will suggest that inhibitig hA3 protein activities may limit acquisition of tumorigenic mutations and drug resistance.